Re: How does the acid-ninhydrin method for proline quantification work?

Date: Mon Jan 21 03:26:29 2008
Posted By: Neil Saunders, Computational biologist
Area of science: Biochemistry
ID: 1200612152.Bc

Message:

Dear Paula,
Thanks for your interesting question. All too often, biochemical protocols are simply presented to us as recipes, with no explanation as to how they work. I find that there are two approaches which can aid your understanding.

Research the history of the protocol. Find a journal article which cites the technique, then try to follow it backwards through the literature to the time that it was first reported. Also, try to find a good review article about the technique. When you do this, you will often find that the answer to "why is it done this way?" is very often "because one person tried it and it worked so we all copy them!"

I find that Google Scholar is an excellent resource for researching articles. Head over there and try a query such as "proline ninhydrin quantitation" and see what it throws up. I have cited some key references at the end of this answer which I found via Google Scholar and hopefully your university has access to these articles. If not, I'm happy to send them to you as PDF files.

Have a good understanding of basic chemistry and spectroscopy. In particular, you should know the Beer-Lambert law [1]. Most biochemical assays involve quite simple chemical reactions between a reagent and a group on the biomolecule (an amino acid side chain in this case). Knowing the structures and chemical properties of the amino acids will help you no end [2].

Let's start with a few words about spectrophotometric assays. They all follow the same basic principle: a compound reacts with the molecule that you want to measure to form a product that absorbs light at a specific wavelength (the product is often coloured). You can then prepare a standard curve by reacting known amounts of the pure molecule with your compound, measuring the absorbance of the resulting solution and plotting absorbance versus concentration. If all goes well the curve will be linear (a straight line). So by measuring the absorbance of solutions containing an unknown concentration of your molecule, you can use the curve to estimate the concentration.

Next, a short history lesson. The use of ninhydrin to quantitate amino acids goes way back to a German chemist named Siegfried Ruhemann, in 1910. He discovered that ninhydrin reacts with primary amine groups to form a compound referred to as Ruhemann's Purple [3]. However, ninhydrin also undergoes many other reactions that form coloured substances. In particular, the reaction with proline is different to that with other amino acids because proline is an imine in which the amine group has cyclised with the side chain [4]. However, since the reaction product of proline and ninhydrin is coloured and absorbs at a specific wavelength, it can be used to quantitate proline in much the same way as other ninhydrin products [5, 6, 7, 8].

Now, the specific parts of your question:

1. Why are plant extracts prepared in 3% sulphosalicylic acid (and not in some kind of buffer)?
First - what's a buffer? It's a substance that maintains the pH of a solution within a certain range. Why are buffers used? To provide an optimum pH for a chemical or biochemical reaction. In this case, we don't need a buffer because we are not performing such a reaction. The role of sulphosalicylic acid is a protein precipitant. It causes large protein molecules to aggregate - they can then be removed by centrifugation, leaving only free amino acids and other small solutes in solution.

2. What happens when we add a mixture of acid-ninhydrin and acetic acid (what is the chemical function of these substances)?
Proline forms different reaction products with ninhydrin, depending on the pH. At neutral pH, a red product is formed with an absorption maximum at ~ 550 nm. At acidic pH, the product is also red but absorbs maximally at ~ 515 nm. Addition of alkali turns the product blue and it now becomes water-soluble. So, the acid is used to generate the required reaction product.
Acidity is also required to maintain ninhydrin in a stable, soluble form, hence the name acid-ninhydrin.

3. Why to boil the mixture for 1h?
Many chemical reactions proceed faster if you raise the temperature (because the frequency of collisions between molecules is increased). In this case, the reaction goes to completion in 30 minutes at 100 °C [5].
Boiling in acid is also likely to hydrolyse molecules that contain proline (such as small peptides) into their constituent amino acids. Do you think that this is relevant in this assay?

4. What does it mean "the reaction mixture was extracted with toluene"?
Toluene is an organic solvent and is immiscible (does not mix) with water [9]. If you add toluene to a water-based solution, shake it up and let it settle, two layers or "phases" will form. Hydrophobic molecules that do not dissolve in water will dissolve in the toluene layer and can be removed. That's what we mean by extraction with toluene.

5. Why is proline the only amino acid present in the toluene phase?
Think about it - proline is not present in the toluene phase. What is present is the reaction product of proline + ninhydrin. As to why this is the only amino acid-ninhydrin product in the toluene phase - you can figure that out logically! It must be the only product that is more soluble in toluene than water.

A question for you: do you really believe that the proline-ninhydrin product is the only compound in the toluene phase, given the complex starting material (whole plant extract)? And if not - do you think it matters with respect to the accurate estimation of proline?

I hope that this helps with your question - don't forget the chemistry in biochemistry!

Neil

References
[1] Beer-Lambert law Wikipedia entry
[2] Amino acids at University of Arizona Biology Department
[3] Ruhemann, S. (1910). Triketohydrindene hydrate. Trans. Chem. Soc. 97: 2025-2031.
[4] Proline at University of Arizona Biology Department
[5] Troll, W. and Lindsley. J. (1955). A photometric method for the determination of proline. J. Biol. Chem. 215: 655-660.
[6] Chinard, F.P. (1952). Photometric estimation of proline and ornithine. J. Biol. Chem. 199: 91-95.
[7] Bates, L.S., Waldren, R.P. and Teare, I.D. (1973). Rapid determination of free proline for water-stress studies. Plant and Soil 39: 205-207.
[8] Friedmann, M. (2004). Applications of the ninhydrin reaction for analysis of amino acids, peptides and proteins to agricultural and biomedical sciences. J. Agric. Food Chem. 52: 385-406.
[9] Toluene InChem datasheet

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